Treating cardiovascular or renal diseases

ABSTRACT

This document provides methods and materials for treating cardiovascular and/or renal diseases. For example, AAV9 vectors designed to express natriuretic polypeptides, nucleic acid molecules encoding natriuretic polypeptides, methods for making AAV9 vectors, and methods for using such vectors or molecules to treat cardiovascular and/or renal diseases are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/436,426, filed Feb. 17, 2017, which is a continuation of U.S.application Ser. No. 14/370,554 (now U.S. Pat. No. 9,611,305), filedJul. 3, 2014, which is a National Stage application under 35 U.S.C. §371 of International Application No. PCT/US2013/020392, having anInternational Filing Date of Jan. 4, 2013, which claims the benefit ofpriority to U.S. Provisional Application Ser. No. 61/584,006, filed onJan. 6, 2012. The disclosures of the prior applications are consideredpart of (and are incorporated by reference in) the disclosure of thisapplication.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numberHL098502 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

TECHNICAL FIELD

This document relates to methods and materials involved in treatingcardiovascular and/or renal diseases. For example, this document relatesto adeno-associated virus serotype 9 (AAV9) vectors designed to expressnatriuretic polypeptides, nucleic acid molecules encoding natriureticpolypeptides, methods for making AAV9 vectors, and methods for usingsuch vectors or molecules to treat cardiovascular and/or renal diseases.

BACKGROUND INFORMATION

Hypertension is a highly common condition that, if not controlled,progresses toward more severe cardiovascular and renal morbidity. Itsmajor clinical phenotype is hypertensive heart disease (HHD), which ischaracterized by diastolic dysfunction, cardiac remodeling, andfibrosis. Over time, diastolic dysfunction evolves into systolicimpairment, which leads to the worsening of overall cardiac function andto increased morbidity and mortality.

SUMMARY

This document provides methods and materials for treating cardiovascularand/or renal diseases. For example, this document provides AAV9 vectorsdesigned to express natriuretic polypeptides, nucleic acid moleculesencoding natriuretic polypeptides, methods for making AAV9 vectors, andmethods for using such vectors or molecules to treat cardiovascularand/or renal diseases. AAV9 was isolated from human tissues and shown tohave serological characteristics distinct from previously describedserotypes (Gao et al., J. Virol., 78:6381-6388 (2004)).

As described herein, AAV9 vectors can be designed to have a nucleic acidsequence that encodes a natriuretic polypeptide such as an atrialnatriuretic polypeptide (ANP), a B-type natriuretic polypeptide (BNP), aC-type natriuretic polypeptide (CNP), or a chimeric natriureticpolypeptide called CDNP. Such AAV9 vectors can be administered to amammal (e.g., a human patient identified as suffering from acardiovascular and/or renal disease) to treat that mammal'scardiovascular and/or renal disease. For example, an AAV9 vectorprovided herein can successfully deliver nucleic acid to cardiac cellsfor expression (e.g., sustained expression) of a natriuretic polypeptidewithout any short- or long-term toxicological effects and any signs oftolerance. In some cases, the sustained cardiac natriuretic polypeptide(e.g., BNP or CDNP) overexpression can reduce blood pressure (BP) andimprove left ventricular (LV) function after a single administration(e.g., a single intravenous injection). In some cases, the AAV9 vectorsprovided herein can be used to reduce or prevent the development ofhypertensive heart disease (HHD). Having the ability to deliver andexpress natriuretic polypeptide in cardiac cells as described herein canallow patients and clinicians to treat cardiovascular and/or renaldiseases in an efficient and effective manner.

In general, one aspect of this document features an AAV9 vectorcomprising, or consisting essentially of, a nucleic acid sequenceencoding a natriuretic polypeptide. The natriuretic polypeptide can be ahuman BNP polypeptide. The natriuretic polypeptide can be a CDNPpolypeptide. The natriuretic polypeptide can be a B-CDNP polypeptide.The natriuretic polypeptide can be a C-CDNP polypeptide.

In another aspect, this document features a composition comprising, orconsisting essentially of, an AAV9 vector in combination with apharmaceutically acceptable delivery vehicle. The AAV9 vector comprises,or consists essentially of, a nucleic acid sequence encoding anatriuretic polypeptide. The natriuretic polypeptide can be a human BNPpolypeptide. The natriuretic polypeptide can be a CDNP polypeptide. Thenatriuretic polypeptide can be a B-CDNP polypeptide. The natriureticpolypeptide can be a C-CDNP polypeptide.

In another aspect, this document features a method for a cardiovascularor renal disease. The method comprises, or consists essentially of,administering a vector or a composition to a mammal. The vector can bean AAV9 vector comprising, or consisting essentially of, a nucleic acidsequence encoding a natriuretic polypeptide, and the composition cancomprise, or consist essentially of, an AAV9 vector in combination witha pharmaceutically acceptable delivery vehicle. The natriureticpolypeptide can be a human BNP polypeptide. The natriuretic polypeptidecan be a CDNP polypeptide. The natriuretic polypeptide can be a B-CDNPpolypeptide. The natriuretic polypeptide can be a C-CDNP polypeptide.The mammal can be a human.

In another aspect, this document features a method for prolongingsurvival time for a mammal with hypertensive heart disease. The methodcomprises, or consists essentially of, administering a vector or acomposition to the mammal. The vector can be an AAV9 vector comprising,or consisting essentially of, a nucleic acid sequence encoding anatriuretic polypeptide, and the composition can comprise, or consistessentially of, an AAV9 vector in combination with a pharmaceuticallyacceptable delivery vehicle. The natriuretic polypeptide can be a humanBNP polypeptide. The natriuretic polypeptide can be a CDNP polypeptide.The natriuretic polypeptide can be a B-CDNP polypeptide. The natriureticpolypeptide can be a C-CDNP polypeptide. The mammal can be a human.

In another aspect, this document features a method for improving cardiacfunction in a mammal with hypertensive heart disease. The methodcomprises, or consists essentially of, administering a vector or acomposition to the mammal under conditions wherein cardiac function isimproved at least five months following the administration. For example,cardiac function can be improved for a period of time extending fromabout five months following the administration to about twelve monthsfollowing the administration, from about five months following theadministration to about ten months following the administration, or fromabout five months following the administration to about six monthsfollowing the administration). The vector can be an AAV9 vectorcomprising, or consisting essentially of, a nucleic acid sequenceencoding a natriuretic polypeptide, and the composition can comprise, orconsist essentially of, an AAV9 vector in combination with apharmaceutically acceptable delivery vehicle. The natriureticpolypeptide can be a human BNP polypeptide. The natriuretic polypeptidecan be a CDNP polypeptide. The natriuretic polypeptide can be a B-CDNPpolypeptide. The natriuretic polypeptide can be a C-CDNP polypeptide.The mammal can be a human.

In another aspect, this document features a method for improving cardiacfunction in a mammal with polycystic kidney disease. The methodcomprises, or consists essentially of, administering a vector or acomposition to the mammal under conditions wherein cardiac function isimproved at least two months following the administration. For example,cardiac function can be improved for a period of time extending fromabout two months following the administration to about twelve monthsfollowing the administration, from about two months following theadministration to about ten months following the administration, or fromabout two months following the administration to about five monthsfollowing the administration). The vector can be an AAV9 vectorcomprising, or consisting essentially of, a nucleic acid sequenceencoding a natriuretic polypeptide, and the composition can comprise, orconsist essentially of, an AAV9 vector in combination with apharmaceutically acceptable delivery vehicle. The natriureticpolypeptide can be a human BNP polypeptide. The natriuretic polypeptidecan be a CDNP polypeptide. The natriuretic polypeptide can be a B-CDNPpolypeptide. The natriuretic polypeptide can be a C-CDNP polypeptide.The mammal can be a human.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting. The details of one ormore embodiments of the invention are set forth in the accompanyingdrawings and the description below. Other features, objects, andadvantages of the invention will be apparent from the description anddrawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A-C. Generation of pre-proBNP-expressing AAV9 vector. (A)Schematic representation of the AAV9 vector encoding rat pre-proBNP. SD;splice donor, SA; splice acceptor, WPRE; woodchuck hepatitis viruspost-transcriptional regulatory elements. CMV, Cytomegalovirus; SD,splice donor; SA, splice acceptor; WPRE, woodchuck hepatitis virusposttranscriptional regulatory element. (B) Verification of proBNPexpression in 293T cells transfected with pAAV-r-proBNP. ImmunoreactiveBNPs in cell lysates and culture supernatants were detected by anti-ratBNP45 antibody. HMW, high molecular weight. (C) Immunostaining of ratBNP in pAAV-r-proBNP-transfected mouse cardiomyocytes (HL1 cells). Whenthe plasmid encoding for the full length of pre-proBNP was introduced inHL1 cells, immunoreactive BNP signals were detected supra-nuclearly andin cytoplasmic secretory vesicles.

FIGS. 2A-D. AAV9 vector facilitates efficient cardiac gene delivery inspontaneously hypertensive rats (SHR). (A) Distribution of luciferaseactivities in firefly luciferase-expressing AAV9 vector-administered SHRorgans was monitored by Xenogen IVIS Living Image. Strong luciferaseexpression in heart demonstrated efficient cardiac gene delivery by AAV9in SHR (n=2) (left panel). Higher magnifications of Luciferase signalswere found in both atria and ventricles (right panels). (B) Detection ofluciferase by immunostaining. Luciferase in the sections of heartventricles were detected by anti-firefly luciferase antibody, confirmingthe efficient cardiac luciferase gene expression upon AAV9vector-mediated gene transfer. (C) No apparent toxicity observed in AAV9vector-administered SHR. Toxicological and pharmacological parameters invector-injected SHR (n=3) were measured at 4 days and 3 weeks aftervector administration. Averages of three rats were shown. *P<0.05 vsrespective untreated controls. WBC, white blood cells; RBC, red bloodcell; HGB, hemoglobin; HCT, hematocrit; PLT, platelets; ALB, albumin;ALP, alkaline phosphatase; ALT, alanine transferase; AMY, amylase; TBIL,total bilirubin; BUN, blood urea nitrogen; PHOS, phosphorus; CRE,creatinine. (D) Sustained BNP expression in the proBNP-expressingvector-administered rats (n=3). The levels of plasma immunoreactive BNPwere measured at 4 days and 3 weeks after vector administration by therat BNP45 ELISA. Error bars indicate ±SD. *P<0.05 vs respectiveuntreated controls.

FIGS. 3A-C. Effects of long-term BNP overexpression in SHR. (A) Noapparent toxicity observed in SHR at 4 months after proBNP-expressingAAV9 vector-administration. Toxicological and pharmacological parametersof the treated and untreated SHR were shown (n=4). No toxicity wasobserved in the rats, while plasma immunoreactive BNP45 wassignificantly elevated in the AAV9 vector-treated group. WBC, whiteblood cells; RBC, red blood cell; HGB, hemoglobin; HCT, hematocrit; PLT,platelets; ALB, albumin; ALP, alkaline phosphatase; ALT, alaninetransferase; AMY, amylase; TBIL, total bilirubin; BUN, blood ureanitrogen; PHOS, phosphorus; CRE, creatinine; GLU, glucose; TP, totalprotein. (B) BP measurements of proBNP-expressing AAV9vector-administered SHR. BP in treated and untreated SHR were measuredby tail-cuff method (n=8). *P<0.05 vs respective untreated controls. (C)M-mode echocardiography of untreated and AAV9-proBNP treated SHR at fourand nine months post AAV9 vector injection. AAV9 vector-treated SHR hadsignificantly improved diastolic functions at four months and bothdiastolic and systolic functions at nine months as compared withuntreated SHR. LVDs, left ventricular end-systolic dimension; LVDd, leftventricular end-diastolic dimension.

FIGS. 4A-D. Effects of long-term BNP overexpression on cardiacremodeling. (A) Intra-arterial measurements of heart rate (HR), systolicblood pressure (SBP), and diastolic blood pressure (DBP) inanesthetized, treated and untreated, SHR are indicated ±SD. (B) Plasmaimmunoreactive BNP45, plasma cGMP, and urinary cGMP are shown (n=4).Error bars indicate ±SD. *P<0.05 vs respective untreated controls. (C)Cross sections were stained by Mason's trichrome staining for musclefibers (red staining) and collagen/fibrosis (blue staining).Representative images of whole section of treated and untreated SHR(upper panels) and higher magnifications of heart images of rats (n=4,lower panels) are shown. (D) Connective tissue deposition was evaluatedas percent blue signals/red signals by KS400 Image Analysis software.The averages of four heart samples in treated and untreated SHR groupsare shown.

FIGS. 5A-C. Effects of non-cardiac BNP overexpression in SHR. (A)Distribution of luciferase activities in firefly luciferase-expressingAAV2 vector-administered SHR organs was monitored by Xenogen LivingImage. Strong luciferase expression was evident in peritoneum by AAV2 inSHR (left panel), while no detectable luciferase expression was observedin heart (right panels). (B) BP measurements of proBNP-expressing AAV2and AAV9 vector-administered SHR. BP in AAV2-treated (n=5), AAV9-treated(n=3), and untreated SHR (n=5) were measured by tail-cuff method. Errorbars indicate ±SD. *P<0.05 vs respective untreated controls. (C) Plasmaimmunoreactive BNP45 and the heart weight/body weight ratios are shown.Error bars indicate ±SD. *P<0.05 vs respective untreated controls.

FIGS. 6A-C. Effects of AAV9 vector-mediated long-term BNP expression innormal Wistar rats. (A) Efficient cardiac transgene expression uponsystemic AAV9 vector-administration. Normal rats were injected byGFP-carrying AAV9 vector. Four weeks after injection, heart sectionswere analyzed for GFP expression. (B) Plasma immunoreactive BNP and theheart weight/body weight ratios are shown. Error bars indicate ±SD.*P<0.05 vs respective untreated controls. (C) Intra-arterialmeasurements of heart rate (HR), systolic blood pressure (SBP), anddiastolic blood pressure (DBP) in anesthetized, treated and untreated,SHR are indicated ±SD.

FIG. 7 contains graphs plotting heart, lung, or kidney weights as apercentage of body weight for untreated SHR or SHR treated with a AAV9viral vector designed to express proBNP or proCDNP.

FIG. 8 contains graphs plotting the levels of CDNP in heart andcirculation, which were measured by an ELISA kit detecting NT-proCNP.

FIG. 9 contains photographs of tissues from spontaneously hypertensiverats 18 months after being exposed to an AAV9 vector designed to expressluciferase. The tissues were assessed for luciferase activity. In bothcases, the heart tissue exhibited high levels of luciferase activity.

FIG. 10 is graph plotting percent survival of 9 month-old SHR rats withestablished hypertensive heart disease that were treated with an AAV9vector designed to express BNP. Controls were comparable rats nottreated with the AAV9 vector.

FIG. 11 is a table demonstrating that sustained cardiac proBNPover-expression by the AAV9 vector improved cardiac function andstructure in mammals with established hypertensive heart disease.

FIG. 12A contains schematics of various AAV vectors that can be designedto express the indicated natriuretic polypeptides. FIG. 12B is aphotograph of a Western blot analysis demonstrating the expression ofCNP from an AAV9-CNP vector and the expression of CDNP from anAAV9-C-CDNP vector.

FIG. 13 is a table demonstrating that sustained cardiac proBNPover-expression by the AAV9 vector improved cardiac function andstructure in mammals with polycystic kidney disease.

FIG. 14 contains amino acid sequences for ANP, pre-pro-ANP, mANP, andpre-pro-mANP as well as a codon optimized nucleic acid sequence thatencodes pre-pro-mANP.

DETAILED DESCRIPTION

This document provides methods and materials for treating cardiovascularand/or renal diseases. For example, this document provides AAV9 vectorsdesigned to express natriuretic polypeptides, nucleic acid moleculesencoding natriuretic polypeptides, methods for making AAV9 vectors, andmethods for using such vectors or molecules to treat cardiovascularand/or renal diseases. In some cases, an AAV8 vector can be used inplace of an AAV9 vector described herein to obtain an AAV8 vectordesigned to express one or more natriuretic polypeptides. Such AAV8vectors can be uses as described herein with respect to AAV9 vectors.

As described herein, an AAV9 vector can be configured to include anucleic acid sequence that encodes a natriuretic polypeptide. Examplesof natriuretic polypeptides that can be expressed using an AAV9 vectoras described herein include, without limitation, ANP (e.g., human ANP),BNP (e.g., human BNP), CNP (e.g., human CNP), CDNP, DNP, mANP, andASBNP. The core amino acid sequence of CDNP can be as follows:GLSKGCFGLKLDRIGSMSGLGCPSLRDPRPNAPSTSA (SEQ ID NO:6). The amino acidsequence for ANP, pre-pro-ANP, mANP, and pre-pro-mANP can be as setforth in FIG. 14. In some cases, the nucleic acid sequence encoding anatriuretic polypeptide can be codon optimized. For example, a codonoptimized nucleic acid sequence designed to encode pre-pro-mANP as setforth in FIG. 14 can be using to make an AAV9 or AAV8 vector providedherein.

In some cases, an AAV9 vector can be configured to include two or moredifferent nucleic acid sequences that encode natriuretic polypeptides.For example, an AAV9 vector can be configured to include a nucleic acidsequence that encodes human BNP and a nucleic acid sequence that encodesCDNP.

In some cases, the one or more natriuretic polypeptides to be expressedusing an AAV9 vector can include the N-terminal region of a naturalnatriuretic polypeptide that includes non-active components of an activenatriuretic polypeptide such as a signal peptide sequence and othersequences that can be involved in polypeptide processing, folding, andstabilization. Examples of such N-terminal regions include, withoutlimitation, those set forth in SEQ ID NO: 1, 4, or 5. In some cases, oneor more of the following sequences can be used as an N-terminal regionof a natriuretic polypeptide to be expressed using an AAV9 vector: BNPsignal peptide +NT-proBNP, CNP signal peptide +NT-proCNP, and ANP signalpeptide +NT-proANP. Examples of amino acid sequences encoding anatriuretic polypeptide that includes such an N-terminal region include,without limitation, those amino acid sequences set forth in SEQ ID NO:3, 7, 8, or 13.

A nucleic acid sequence (e.g., a nucleic acid sequence optimized forhuman codon usage) encoding a natriuretic polypeptide described hereincan be inserted into any appropriate AAV9 viral vector. For example, anucleic acid sequence encoding a human CDNP can be inserted into an AAV9vector having a nucleic acid sequence as set forth in GenBank® AccessionNo. AY530557 (GI No. 46487760), JA400113.1 (GI No. 346220229), JA232063(GI No. 330731135), JA231827 (GI No. 330729561), or

JA062576 (GI No. 328343515). In some cases, an AAV9 vector can have thesequence as described elsewhere. See, e.g., WO2003/052052, U.S. PatentApplication Publication No. 20110236353, EP2345731, EP2292780,EP2292779, or EP2298926. In some cases, an AAV vector (e.g., AAV9 orAAV8 vectors) can be designed to express BNP, CNP, or CDNP as set forthin FIG. 12A.

In some cases, a promoter sequence can be operably linked to a nucleicacid sequence that encodes a natriuretic polypeptide (e.g., BNP,pre-proBNP, CDNP, B-CDNP, or C-CDNP) to drive expression of thenatriuretic polypeptide. Examples of such promoter sequences include,without limitation, CMV, El1alpha, BNP, CNP, ANP, MYH6 , and MYH7promoters. In some cases, a promoter sequence that is active underconditions of elevated blood pressure with minimal, or no, activityunder conditions of normal or low blood pressure can be operably linkedto a nucleic acid sequence that encodes a natriuretic polypeptide (e.g.,BNP, pre-proBNP, CDNP, B-CDNP, or C-CDNP) to drive expression of thenatriuretic polypeptide under conditions of elevated blood pressure.Examples of such blood pressure sensitive promoter sequences include,without limitation, BNP and ANP promoters.

In one aspect, this document provides AAV9 vectors containing a nucleicacid sequence that encodes a natriuretic polypeptide. Such AAV9 vectorscan infect cardiac cells and direct the expression of the natriureticpolypeptide by the infected cells.

Any appropriate method can be used to insert nucleic acid (e.g., nucleicacid encoding a natriuretic polypeptide) into the genome of an AAV9vector. For example, standard molecule biology techniques such asrestriction enzyme cutting, ligations, and homologous recombination canbe used to insert nucleic acid into the genome of an AAV9 vector. Anyappropriate method can be used to identify AAV9 vectors containing anucleic acid molecule that encodes a natriuretic polypeptide. Suchmethods include, without limitation, PCR and nucleic acid hybridizationtechniques such as Northern and Southern analysis. In some cases,immunohistochemistry and biochemical techniques can be used to determineif an AAV9 vector contains a particular nucleic acid molecule bydetecting the expression of a polypeptide encoded by that particularnucleic acid molecule.

In another aspect, this document provides nucleic acid molecules thatencode a natriuretic polypeptide. For example, a nucleic acid moleculeprovided herein can be a single nucleic acid molecule that encodes theN-terminal region of a natural natriuretic polypeptide that includes oneor more non-active components of an active natriuretic polypeptide suchas a signal peptide sequence and other sequences that can be involved inpolypeptide processing, folding, and stabilization upstream of an activecomponent of a natriuretic polypeptide. In some cases, such a nucleicacid molecule can have a nucleic acid sequence that encodes anatriuretic polypeptide set forth in SEQ ID NO: 3, 7, 8, or 13.

In some cases, a nucleic acid molecule provided herein can include apromoter sequence operably linked to the nucleic acid sequence encodinga natriuretic polypeptide. For example, a nucleic acid molecule providedherein can include a promoter sequence that is active under conditionsof elevated blood pressure operably linked to a nucleic acid sequenceencoding a natriuretic polypeptide (e.g., CDNP).

The term “nucleic acid” as used herein encompasses both RNA and DNA,including cDNA, genomic DNA, and synthetic (e.g., chemicallysynthesized) DNA. A nucleic acid can be double-stranded orsingle-stranded. A single-stranded nucleic acid can be the sense strandor the antisense strand. In addition, a nucleic acid can be circular orlinear.

This document also provides methods for treating cardiovascular and/orrenal diseases (e.g., to reduce blood pressure, cardiomyocytehypertrophy, cardiac fibrosis, or renal fibrosis, or to improve systolicand diastolic dysfunctions). For example, an AAV9 vector provided hereincan be administered to a mammal having a cardiovascular and/or renaldisease to reduce blood pressure within the mammal. An AAV9 vectorprovided herein can be produced in human cell lines, such as 293T cells,or other types of cells such as insect cells, which can be concentratedtypically by at least 100-fold, or even by as much as 5,000- to10,000-fold, through ultracentrifugation. A viral titer typically isassayed by measuring the viral vector copy numbers inconcentrated/purified vector preparations.

An AAV9 vector provided herein can be administered to a patient (e.g.,human patient) by, for example, direct injection into a group of cardiaccells or intravenous delivery to cardiac cells. An AAV9 vector providedherein can be administered to a patient in a biologically compatiblesolution or a pharmaceutically acceptable delivery vehicle such assaline, by administration either directly into a group of cardiac cellsor systemically (e.g., intravenously). Suitable pharmaceuticalformulations depend in part upon the use and the route of entry. Suchforms should not prevent the composition or formulation from reaching atarget cell (i.e., a cell to which the virus is desired to be deliveredto) or from exerting its effect. For example, pharmacologicalcompositions injected into the blood stream should be soluble.

While dosages administered will vary from patient to patient, aneffective dose can be determined by setting as a lower limit theconcentration of virus proven to be safe and escalating to higher dosesof up to 10¹³ vector genome copies (vg)/kg, while monitoring for aresponse (e.g., a reduction in blood pressure) along with the presenceof any deleterious side effects. Escalating dose studies can be used toobtain a desired effect for a given viral treatment.

An AAV9 vector provided herein can be delivered in a dose ranging from,for example, about 10³ vg/kg to about 10¹³ vg/kg. A therapeuticallyeffective dose can be provided in repeated doses. Repeat dosing isappropriate in cases in which observations of clinical symptoms ormonitoring assays indicate that the degree of viral activity (e.g.,natriuretic polypeptide expression) is declining. Repeat doses can beadministered by the same route as initially used or by another route. Atherapeutically effective dose can be delivered in several discretedoses (e.g., days, weeks, months, or years apart).

An AAV9 vector provided herein can be directly administered to cardiaccells. For example, a virus can be injected directly into heart tissue.In some cases, ultrasound guidance can be used in such a method. In somecases, an AAV9 vector provided herein can be delivered systemically. Forexample, systemic delivery can be achieved intravenously via injection.The course of therapy with an AAV9 vector provided herein can bemonitored by evaluating changes in clinical symptoms.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Long-Term Cardiac proBNP Gene Delivery Prevents theDevelopment of Hypertensive Heart Disease in Spontaneously HypertensiveRats SHR and Wistar Rats

Four week-old SHR and five week-old Wistar rats were purchased fromCharles River. SHR served as a model of progressive HHD. Strains ofrats, number of animals, treatment, and duration of treatment in eachexperiment was summarized in Table 1. All animal studies were approvedby the Institutional Animal Care and Use Committee.

TABLE 1 Summary of the rats used. Duration Study Strain Treatment Number(weeks) Cardiac delivery SHR AAV9-Luc 2 3 Toxicology SHR Untreated 3 3SHR AAV9-Luc 3 3 SHR AAV9-BNP 3 3 Pharmacokinetics/ SHR Untreated 8 40dynamics SHR AAV9-BNP 8 40 Non-cardiac SHR Untreated 5 8 delivery SHRAAV2-BNP 5 8 SHR AAV9-BNP 3 8 Normotensive Wistar AAV9-GFP 6 4 rat studyWistar AAV9-BNP 6 4 SHR, spontaneously hypertensive rats; Luc,luciferase; GFP, green fluorescent protein.

Cell Culture

HEK 293T cells were maintained in Dulbecco's modified Eagle's mediumsupplemented with 10% calf serum, 50 U/mL penicillin, and 50 μg/mLstreptomycin. A murine atrial cardiomyocyte cell line, HL-11, wasobtained from Dr. William C. Claycomb (Louisiana State UniversityMedical Center, New Orleans) and cultured in Claycomb's medium with 10%FBS, 100 μM norepinephrine, and 4 mM L-glutamine on 0.02%gelatin/fibronectin-coated flasks or plates.

Transfection, Immunoblotting and Immunostaining

Fugene6 (Roche) was used for transfection. For immunoblotting,immuno-reactive rat BNPs were detected using rabbit anti-rat BNP1-45antibody (AssayPro) and HRP-conjugated anti-rabbit IgG antibody.Immunostaining of immuno-reactive rat BNP was performed using the sameanti-rat BNP1-45 antibody and FITC-conjugated anti-rabbit IgG antibody.

IVIS Imaging

The cardiac luciferase expression was monitored by Xenogen IVISbiophotonic imaging machine. Upon luciferin administration through IP,anesthetized rats were euthanized, and the organs were harvestedimmediately. Harvested tissues were placed on the 10-cm plates on theimaging chamber, and a background photo of the tissues and a coloroverlay of the emitted photon data were obtained.

Toxicological and Pharmacological Tests

For toxicological and pharmacological tests, hematological parameters(VetScan HM2 Hematology System; 504 blood in EDTA for WBC counts, WBChistogram, Hb, Hct, MCV, MCH, MCHC, RDW, graphic RBC histogram, PLTcount, MPV, PCT, PDW and Graphic platelet histogram) and chemistry(VetScan Classic; 100 μL blood in lithium heparin; ALB, ALP, ALT, AMY,BUN, CA++, CRE, GLOB, GLU, K+, Na+, PHOS, TBIL, TP) were measured.

Plasmids

The codon-optimized rat pre-proBNP was synthesized by GenScript, andcloned into a lentiviral vector, pSIN-CSGWdlNotI. The BamHI-Xhol shortfragment, which contained rat pre-proBNP and WPRE post-transcriptionalregulatory element, was then cloned into the mammalian expressionplasmid, pAAV-MCS (Stratagene), resulting in pAAV-rat-pre-proBNP.

AAV9 and AAV2 Vectors

The AAV9 vector stocks were produced in human 293T cells using thehelper-free transfection method according to the manufacturer's protocol(Stratagene). For AAV9 vector production, AAV9 capsid-expressing plasmidpRep2Cap9 (obtained from Dr. Hiroyuki Nakai) was used, while AAV2 vectorwas made with the AAV2 capsid-expressing plasmid, pAAV-RC (Stratagene).Firefly luciferase-, humanized recombinant green fluorescent protein(GFP)-, or rat proBNP-encoding AAV genome constructs were packaged.Three days after transfection, AAV9 vector-producing 293T cells wereharvested for vector purification. The cells were lysed by freeze andthaw cycling, followed by ultracentrifuge concentration (62,500 rpm for2 hours) through Optiprep Density Gradient Medium (Sigma). The resultingAAV9 vectors were desalted and further concentrated using AmiconUltra-15 100k filtration (Amicon). The titers (genomic copy numbers/mL)of concentrated AAV9 vector stocks were determined by quantitative PCRusing plasmid DNA standards, AAV genomic sequence-specific primers, anda fluorescent probe.

Non-Invasive Tail Blood Pressure Measurement

The blood pressure (BP) of conscious rats was measured by the CODAHigh-Throughput Non-Invasive Tail BP System (Kent Scientific).

Echocardiography (ECHO) for Non-Invasive Assessment of VentricularFunction and Structure

To evaluate cardiac function and structure, both standard ECHO andTwo-Dimensional Speckle-Derived Strain ECHO (2DSE) examinations wereperformed at four and nine months post injections in the BNP-treated andthe untreated SHR. Standard ECHO and 2DSE also were performed in normalWistar rats at 4 weeks after AAV9 injections. All ECHO examinations wereperformed by a skilled sonographer blinded to the treatment.

Standard ECHO was performed as follows. After removing chest hair,ultrasonic scans was performed in all rats in supine position using aVivid 7 system (GE Healthcare, Milwaukee, Wis.) equipped with a 10Sultrasound probe (11.5 MHz) with ECG monitoring. M-mode images and grayscale 2D images (300-350 frames/sec) of the parasternal long-axis andmid-LV was recorded for off-line analysis. LV end-diastolic (LVDd) andend-systolic (LVDs) dimensions and septal diastolic (SWTd) and posteriorwall diastolic (PWTd) and systolic (PWTs) thicknesses were measured fromM-mode images. LV mass was calculated according to uncorrected cubeassumptions as LV mass=1.055×[(LVDd+SWTd+PWTd)³−(LVDd)³], where 1.055 isthe specific gravity of myocardium. LV mass was corrected for bodyweight (LVMi) for analysis. End-systolic (ESV), end-diastolic and strokevolumes (SV), and ejection fraction (EF) was calculated using theTeichholz formula: LV volume=7×[(LVDd)³/(2.4+LVDd)]. Relative wallthickness (RWT) was calculated as RWT=(SWTd +PWTd)/LVDd. All parametersrepresented the average of three beats.

2DSE was performed as follows. Using EchoPAC software (EchoPAC PC—2Dstrain, BTO 6.0.0, GE Healthcare, Milwaukee, Wis.), which included ahigh resolution speckle tracking analysis library for off-line analysis,endocardial border was carefully manually traced at end-systole in LVshort-axis views at the middle level (i.e., at the level of papillarymuscles). Ideal width of circular region of interest was chosen in orderto include the entire myocardial wall. Speckle tracking was performed bythe software and global strain, and circumferential strain rateparameters were measured computing the mean of the six middle LVsegments. The analysis included peak circumferential systolic strains(sS) and strain rates (sSR) for evaluation of myocardial systolicfunction and peak early circumferential strain rates (dSR-E) forevaluation of myocardial diastolic function. All parameters representedthe average of three beats. Using standard ECHO and 2DSE, significantimprovement were detected in both systolic and diastolic function in arat model of cardiac dysfunction when compared to the untreated SHR.

Acute Experiment Procedure

Rats for the acute protocol were anesthetized with isoflurane (1.5% inoxygen). Placement of PE-50 tubing into the carotid artery for BPmonitoring and blood sampling were performed. A portion of the neck skinwas removed, and the carotid artery were isolated and cleared. A cut wasmade with micro-scissors, and a PE-50 tubing was introduced into thevessel for direct BP monitoring. Blood was drawn to evaluatetoxicological reactions in AAV9-BNP transduced rats and to measure BNPand cGMP. At the end of the experiments, rat organs were harvested forfurther analysis.

Masson's Trichrome Staining

The sections of frozen cardiac samples were assessed by Trichromestaining for collagen contents. Percent blue signals were analyzed byKS400 Image Analysis Software (version 3.0, Zeiss).

Sample Size and Statistical Analysis

Groups were compared by unpaired t-test, and changes within-groups wereassessed by paired t-test. Comparisons of BP values between groups wereperformed by two-way ANOVA for repeated measurements. Data wereexpressed as mean ±SD. Significance was accepted for p<0.05.

Results

In Vitro Expression and Localization of proBNP

After successfully engineering AAV9 encoding for the rat pre-proBNP,which included the signal peptide (SP), NT-proBNP, and BNP1-45 domains(FIG. 1A), the protein expression of BNP was verified in 293T cells.Non-glycosylated proBNP (10 kDa) and high molecular weight (HMW, 12-24kDa) glycosylated proBNP were detected in the cell lysates by Westernblotting analysis. Of note, HMW forms of BNP were predominantly secreted(FIG. 1B). When the pre-proBNP was expressed in mouse cardiomyocytes(HL-1 cells obtained from Dr. William C. Claycomb) and analyzed byimmunostaining with an anti-rat BNP1-45 antibody, clear supranuclearlocalization of immunoreactive BNP (red staining) as well as discretecytoplasmic body signals (red staining) were detected (FIG. 1C).

In Vivo Cardiac Specific Tropism of AAV9 Vector-Mediated Gene Transfer

The rat pre-proBNP- or firefly luciferase-expressing vectors werepackaged in AAV9 capsid, and the influence of AAV9 vector-mediated genedelivery was examined in SHR. Four week-old SHR (n=2) were used. Threeweeks after tail intravenous injection of AAV9 carrying luciferase (10¹²genome copy/animal), the tissue specificity of the AAV9 vector wasdetermined by luciferase expression in the SHR, which demonstrated highlevels of luciferase expression in myocardium (FIG. 2A-B). To confirmluciferase expression in the heart, the heart section was stained withanti-luciferase antibody, and the signals were detected predominately inthe cardiomyocytes (FIG. 2B). When AAV9-luciferase (n=3) andAAV9-pre-proBNP (n=3) were injected and the acute (4 days) and chronic(3 weeks) toxicological responses were compared to those of theuntreated SHR (n=3), no notable toxicity was observed among these threegroups of rats (FIG. 2B). However, plasma BNP, by rat BNP1-45 ELISA, wassignificantly higher in the AAV9-pre-proBNP-treated SHR compared withuntreated SHR both at four days and three weeks after injections (FIG.2D), thus confirming the sustained BNP expression upon AAV9vector-mediated gene delivery.

Effects of Sustained proBNP Expression in SHR

The effects of sustained proBNP expression in SHR through cardiac proBNPdelivery by AAV9 vector were monitored. Four months afterAAV9-pre-proBNP injections in SHR, there was no toxicological reactioncompared to untreated SHR. Importantly, plasma immune reactive BNP wassignificantly higher in the AAV9-pre-proBNP-treated group compared withthe untreated SHR (FIG. 3A). Tail cuff BP measurements indicatedsignificant reduction in SBP, DBP, and MAP in theAAV9-pre-proBNP-treated SHR as compared with untreated SHR. Indeed, inthe AAV9-pre-proBNP, SBP was significantly reduced one month afterinjection and followed by a reduction in both DBP and MAP at two monthspost-injection as compared with the untreated SHR. These reductions inSBP, DBP, and MAP in conscious rats remained throughout the nine monthstudy (FIG. 3B).

Echocardiographic parameters in untreated SHR and in AAV9 pre-proBNPtreated SHR were summarized in Table 2. While no difference was detectedin HR between AAV9 pre-proBNP treated and untreated SHR both at four andnine months post injection, echo analysis indicated a significantimprovement of diastolic function at four and nine months as well assystolic function at nine months post injection in AAV9 pre-proBNPtreated SHR as compared with untreated SHR (FIG. 3C). Of note, EF, PWTd,LVDd, and dSR-E circumferential were improved, and LVMi was lower atnine months in the AAV9 pre-proBNP treated SHR even when compared tofour months untreated SHR (Table 2).

TABLE 2 Echocardiographic parameters in untreated (n = 8) and BNPtreated SHR (n = 8). 4 Month p.i. 9 Months p.i. Untreated BNP TreatedUntreated BNP Treated HR    403 ± 27.3    392 ± 22.3    381 ± 25.2   393 ± 13 SWTd   2.09 ± 0.1   1.86 ± 0.1*   2.71 ± 0.1†   2.17 ± 0.2*†PWTd   2.03 ± 0.1   1.86 ± 0.2*   2.16 ± 0.3   1.87 ± 0.1*‡ LVDd   6.77± 0.2   6.71 ± 0.1   7.56 ± 0.6†   7.57 ± 0.4†‡ LVDs   3.83 ± 0.4   3.47± 0.1*   4.66 ± 0.6†   3.96 ± 0.3*† Ejection Fraction     80 ± 4.1    85 ± 1.8*     74 ± 4.5†     83 ± 2.1*†‡ LV Mass Index   0.44 ± 0.01   0.4 ± 0.02*   0.49 ± 0.01    0.4 ± 0.01*†‡ sSR Circumferential  −4.6± 0.7 −4.75 ± 0.6 −3.74 ± 0.4† −5.04 ± 0.4* dSR-E Circumferential   2.41± 0.8   4.07 ± 1.5*   2.09 ± 0.8   3.25 ± 0.9*‡ sSR-Radial   7.17 ± 1.1  6.77 ± 0.8   6.34 ± 1.6   8.13 ± 1.9*† dSR-E Radial −3.29 ± 1.2 −5.72± 2.5* −2.41 ± 1.3 −4.57 ± 2.1*† *P < 0.05 vs respective Untreated; †P <0.05 vs four months within group; ‡P < 0.05 between nine monthsBNP-treated and four months Untreated. HR, heart rate; SWTd, septal wallthickness at end diastole; PWTd, posterior wall thickness at enddiastole; LVDd, left ventricular end-diastolic dimension; LVDs, leftventricular end-systolic dimension; sSR, systolic strain rate; dSR,diastolic strain rate.

At nine months post-injection, four rats per group were sacrificed foracute experiments. Direct intra-carotid systolic and diastolic bloodpressure was reduced in the AAV9 pre-proBNP treated anesthetized SHR(FIG. 4A), while no significant differences were found in weights andheart rates between treated and untreated rats. The heart weightcorrected for the body weight was significantly reduced in theBNP-treated as compared with the control SHR (0.37±0.01 vs 0.43±0.02,respectively, p<0.05). Plasma BNP was higher in the BNP-treated ascompared with the control SHR (FIG. 4B). Although plasma cGMP was notdifferent, urinary cGMP was greater in the BNP-treated as compared withthe control SHR (FIG. 4B). Connective tissue (assessed by Mason'strichrome staining) tended to increase in heart sections of untreatedSHR as compared with AAV9-preproBNP treated SHR (FIG. 4C-D).

Non-Cardiac BNP-Transduction by AAV2 Vector

The effects of non-cardiac proBNP gene delivery on BP, plasma BNPlevels, and heart weight in SHR were assessed. For non-cardiac genedelivery, conventional AAV2 vectors were administered throughintra-peritoneal injection. One month after administration of AAV2vector carrying luciferase, high levels of luciferase expression werefound in peritoneum, but not in heart (FIG. 5A), confirming efficient,but non-cardiac, gene delivery by AAV2 vector. To assess the influenceof non-cardiac proBNP gene delivery, SHR were injected withpre-proBNP-carrying AAV2 vector and compared with untreated (n=5) andAAV9-pre-proBNP vector-administered rats (n=3). Tail cuff BPmeasurements indicated that AAV2-pre-proBNP administration had nosignificant effects on SBP and DBP. In contrast, SBP was significantlyreduced five and eight weeks after injection of the AAV9-pre-proBNPvector (FIG. 5B). The pre-proBNP gene delivery by AAV9, but not AAV2,showed significantly higher plasma level of BNP (FIG. 5C) andsignificant reduction in the heart weight/body weight ratio (FIG. 5C),suggesting the requirement of cardiac pre-proBNP delivery for efficientBNP release and the anti-hypertrophic effects of BNP in SHR.

Effects of Sustained proBNP Expression in Normotensive Rats

To investigate whether the beneficial effects on both cardiac structureand function observed in the BNP-treated SHR were mainly due to thesustained BP effects, AAV9 encoding for GFP or pre-proBNP was injectedin normal Wistar rats (n=12). Six rats per group underwentechocardiographic examination 4 weeks after injections and weresacrificed for acute experiments thereafter. Normal rats treated withAAV9 carrying GFP (n=6) showed wide spread GFP expression incardiomyocytes 4 weeks after injection, further confirming the cardiactransduction of the AAV9 vector (FIG. 6A). Echocardiographic examinationby strain analysis demonstrated that at 4 weeks post injection,AAV9-pre-proBNP treated normal rats (n=6) had a significantly improvedsystolic function compared with AAV9-GFP as indicated by a thinner SWTd,and higher sSR circumferential, while LVMi was only slightly reduced(p=0.07, NS) (Table 3). AAV9-pre-proBNP treated normal rats hadsignificantly higher plasma level of BNP compared to the GFP-controlrats (FIG. 6B). Although direct intra-carotid BP measurement foundsimilar SBP, DBP, and MAP between the two groups, the heart weightcorrected for the body weight was significantly reduced in theBNP-treated as compared with the GFP-control rats (FIGS. 6B and 6C).

TABLE 3 Echocardiographic parameters in control (n = 6) and BNP treated(n = 6) normal rats. 4 Weeks p.i. Controls BNP Treated HR    407 ± 27.2   408 ± 24.5 SWTd   2.03 ± 0.15   1.80 ± 0.11* PWTd   1.66 ± 0.09  1.77 ± 0.19 LVDd    7.0 + 0.67    7.1 ± 0.53 LVDs   3.84 ± 0.67   3.94± 0.37 Ejection Fraction     83 ± 1.72     83 ± 4.17 LV Mass Index  0.35 ± 0.01   0.33 ± 0.01 sSR −5.37 ± 0.5 −6.54 ± 0.8* CircumferentialdSR-E   5.09 ± 0.7   5.98 ± 1.0 Circumferential sSR-Radial   7.63 ± 1.5  9.08 ± 1.7 dSR-E Radial −5.59 ± 1.3 −7.84 ± 2.7 *P < 0.05 vsrespective Controls. HR, heart rate; SWTd, septal wall thickness at enddiastole; PWTd, posterior wall thickness at end diastole; LVDd, leftventricular end-diastolic dimension; LVDs, left ventricular end-systolicdimension; sSR, systolic strain rate; dSR, diastolic strain rate.

These results demonstrate successful in vivo cardiomyocyte transductionvia a AAV9 vector that facilitated sustained cardiac proBNPoverexpression. Long-term proBNP delivery led to reduced BP and improvedLV function and structure in an HHD rat model without any short- orlong-term toxicological adverse effects or development of tolerance.Although long-term proBNP delivery improved both systolic and diastolicfunction, the effect on diastolic performance was more remarkable andpreceded the improvement in systolic function in this HHD model.Importantly, the effects on cardiac structure and function occurredindependently of BP lowering effects in normal Wistar rats.

These results also demonstrated that rat BNP is released frompre-proBNP-expressing 293T cells as a HMW form.

In addition, rat proBNP overexpression was associated with significantand sustained BP reduction in SHR. Indeed, SBP, DBP and MAP were lowerin the BNP-treated as compared with the control SHR from two months upto nine months post-AAV9 injection. This reduction of BP was rathermodest and occurred without changes in HR. Of note, BP was reduced atthe vector dose used in the current study, while it did not completelynormalized BP, which remained elevated throughout the period ofobservation. It is possible that a higher vector dose would result in amore profound BP reduction. Although the use of telemetry would havehelped in better assessing BP changes, a significant BP lowering effectof BNP was confirmed in unconscious BNP-treated SHR compared to theuntreated SHR via direct intra-arterial BP measurements at the time ofthe acute experiments (9 months).

Plasma immunoreactive rat BNP45 was elevated in the BNP-treated ascompared with the control SHR at four days, three weeks, and four andnine months post-injection, confirming a sustained overexpression of BNPin the heart. At nine months post-injection, plasma cGMP was notdifferent between the BNP-treated and the control SHR. In contrast,urinary cGMP was increased in the BNP-treated as compared with thecontrol SHR. Thus, the lack of elevation of plasma cGMP may be explainedby the increased urinary cGMP excretion.

Chronic overexpression of proBNP prevented the development of HHD, whichbegan at four weeks of age in the SHR. Indeed, AAV9 induced proBNPproduction resulted in a sustained and significant reduction (up to ninemonths) of SBP and DBP. Thorough echo analysis demonstrated asignificant improvement of diastolic function at four months posttransfection in the BNP-treated group as compared with the untreatedSHR. Importantly, at nine months, untreated SHR also developed signs ofimpaired systolic function which was prevented in the BNP-treated SHR.Of note, global cardiac function and remodeling were not only improvedin the BNP-treated SHR compared to untreated SHR of the same age butalso, BNP-treated SHR at nine months showed improved diastolic functionand reduced cardiac hypertrophy even when compared with the untreatedSHR at four months of age. This finding further supports the beneficialrole of BNP in preventing cardiac dysfunction and remodeling. Of note,all these favorable actions occurred without signs of any short- orlong-term toxicological side effects and BNP maintained its biologicalactions up to nine months post injection without developing tolerance.It should be noted that, although sustained, the BP reduction wasminimal, thus further studies are required to address the pathogenicrole of BNP in hypertension.

In SHR transduced with non-cardiac AAV2 vector, no increase in plasmaimmunoreactive BNP was observed. This could be due to a less efficientintracellular processing and/or release of rat BNP in non-cardiac cells.Furthermore, in these AAV2-transduced SHR, changes in heart weightcompared to untreated controls were not observed. The work describedherein used a comparable vector dose for both the AAV2 and AAV9 studies.

The work was extended to normal rats to investigate theanti-hypertrophic actions of BNP overexpression in the absence ofhypertension. In this model, age induced systolic impairment wassignificantly ameliorated in the BNP-treated rats by echo strainanalysis at 4 weeks. Also, cardiac mass was reduced in the BNP-treatedrats as compared with the controls and heart weight/body weight wassignificantly lower in the BNP-treated rats as compare to the controls.Importantly, the improved cardiac function and the reduced cardiac masswere observed after 4 weeks post injections of the AAV9 vector andoccurred despite any difference in BP (measured directly intra-carotid)between the BNP-treated and the control group.

Taken together, the results provided herein demonstrate that the use ofchronic supplementation of the cardiorenal protective hormone BNP can beemployed in hypertension to prevent the progression toward more severestages of HHD and the onset of heart and renal failure. Instead of oraldelivery, a gene transfection strategy was used to facilitate a ninemonth delivery of bioactive BNP with single intravenous injection of theAAV9 vector. As indicated herein, chronic overexpression of BNP in SHRreduced BP, decreased LVH, tended to reduce fibrosis, and improvedsystolic and diastolic function.

In summary, the results provided herein demonstrate the successfulcardiac delivery of the AAV9 vector, which mediated sustained cardiacproBNP overexpression without any short- or long-term toxicologicaleffects and any signs of tolerance. Importantly, sustained cardiac BNPoverexpression reduced BP and improved LV function in a model ofprogressive HHD after a single intravenous injection. Although long-termproBNP delivery improved both systolic and diastolic function, theeffect on diastolic performance was more remarkable and appeared earlierduring the development of HHD. Ultimately, sustained overexpression ofBNP in SHR prevented the development of HHD as nine month oldBNP-treated SHR had a significantly improved cardiac function andstructure even when compared with four month old untreated SHR.Non-cardiac BNP-overexpression was not associated with increase inplasma BNP, changes in BP, and reduced heart weight. The direct cardiaceffects of overexpressed BNP seem to be, at least in part, independentof BP lowering action as indicated by the improved systolic function andreduced heart weight in the normotensives rats despite no changes in BP.

In addition, an AAV9 vector designed to express luciferase achievedlong-term cardiac luciferase expression in SHR rats at least for 18months (FIG. 9). Generation of AAV9 vectors expressing CNP and CDNP asset forth in FIG. 12A was verified by Western blotting (FIG. 12B) usingan anti-CNP antibody.

Example 2 Use of Natriuretic Polypeptides to Reduce Heart Volume

To deliver rat BNP-45 to rat cardiac cells, a cardio-tropic AAV9 vectorwas designed to express a rat proBNP polypeptide having a rat N-terminusproBNP amino acid sequence (called an NT-proBNP region) upstream of arat BNP-45 amino acid sequence. The amino acid sequence for the ratNT-proBNP region with its signal peptide of the rat proBNP polypeptidewasMDLQKVLPQMILLLLFLNLSPLGGHSHPLGSPSQSPEQSTMQKLLELIREKSEEMAQRQLSKDQGPTKELLKRVLR(SEQ ID NO:1). The amino acid sequence for the rat BNP-45 of the ratproBNP polypeptide was SQDSAFRIQERLRNSKMAHSSSCFGQKIDRIGAVSRLGCDGLRLF(SEQ ID NO:2). The amino acid sequence for the rat pre-proBNPpolypeptide wasMDLQKVLPQMILLLLFLNLSPLGGHSHPLGSPSQSPEQSTMQKLLELIREKSEEMAQRQLSKDQGPTKELLKRVLRSQDSAFRIQERLRNSKMAHSSSCFGQKIDRIGAVSRLGCDGLRLF (SEQ ID NO:3).

To deliver CDNP to rat cardiac cells, two cardio-tropic AAV9 vectorswere designed. One AAV9 vector was designed to express a polypeptidedesignated B-CDNP, and the other AAV9 vector was designed to express apolypeptide designated C-CDNP. The B-CDNP polypeptide included a humanN-terminus proBNP amino acid sequence (called an NT-proBNP region)upstream of the CDNP amino acid sequence, while the C-CDNP polypeptideincluded a human N-terminus proCNP amino acid sequence (called anNT-proCNP region) with its signal peptide upstream of the CDNP aminoacid sequence. The amino acid sequence for human NT-proBNP region withits signal peptide of the B-CDNP polypeptide was with its signal peptideMDPQTAPSRALLLLLFLHLAFLGGRSHPLGSPGSASDLETSGLQEQRNHLQGKLSELQVEQTSLEPLQESPRPTGVWKSREVATEGIRGHRKMVLYTLRAPR (SEQ ID NO:4), and the amino acid sequencefor the rat NT-proCNP region with its signal peptide of the C-CDNPpolypeptide wasMHLSQLLACALLLTLLSLRPSEAKPGAPPKVPRTPPAEELAEPQAAGGGGKKGDKAPGGGGANLKGDRSRLLRDLRVDTKSRAAWARLLGEHPNARKYKGANKK (SEQ ID NO:5). The amino acid sequence for CDNP of the B-CDNPand C-CDNP polypeptides was GLSKGCFGLKLDRIGSMSGLGCPSLRDPRPNAPSTSA (SEQID NO:6). The amino acid sequence for B-CDNP polypeptide wasMDPQTAPSRALLLLLFLHLAFLGGRSHPLGSPGSASDLETSGLQEQRNHLQGKLSELQVEQTSLEPLQESPRPTGVWKSREVATEGIRGHRKMVLYTLRAPRGLSKGCFGLKLDRIGSMSGLGCPSLRDPRPNAPSTSA (SEQ ID NO:7), and the amino acid sequence forC-CDNP polypeptide wasMHLSQLLACALLLTLLSLRPSEAKPGAPPKVPRTPPAEELAEPQAAGGGQKKGDKAPGGGGANLKGDRSRLLRDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGCPSLRDPRPNAPSTSA (SEQ ID NO:8).

The nucleic acid sequences encoding B-CDNP and C-CDNP werecodon-optimized for expression in human cells. The nucleic acid sequenceencoding B-CDNP was as follows5′-ATGGACCCACAGACAGCTCCCAGTAGGGCTTTGCTTCTTTTGCTTTTCCTGCACCTGGCTTTTCTGGGCGGACGATCCCATCCACTGGGTAGCCCTGGCTCCGCCTCAGATCTGGAGACTAGTGGACTGCAGGAGCAGCGCAATCACTTGCAGGGCAAACTGTCCGAGCTGCAGGTGGAACAAACGAGCCTCGAGCCCCTGCAGGAGAGCCCTAGACCTACCGGGGTGTGGAAGTCTCGAGAGGTAGCGACAGAAGGCATTAGAGGGCACAGGAAGATGGTACTGTATACTCTGAGGGCCCCAAGGGGACTGAGCAAGGGCTGTTTTGGCCTGAAGCTGGATCGGATTGGCAGCATGTCCGGCCTGGGCTGCCCTTCCCTGCGGGACCCACGGCCAAATGCCCCC TCCACCAGCGCCTAA-3′(SEQ ID NO:9), while the nucleic acid sequence encoding C-CDNP was asfollows5′-ATGCATCTGTCCCAACTGCTGGCTTGTGCTCTCCTGCTGACTCTGCTGAGCCTCCGGCCTAGCGAGGCCAAGCCTGGAGCACCACCTAAGGTCCCCAGGACTCCTCCAGCCGAAGAACTGGCTGAGCCTCAGGCTGCCGGGGGCGGGCAGAAGAAAGGAGACAAAGCCCCTGGAGGGGGCGGGGCTAATCTCAAGGGCGATAGGTCCAGACTGCTGAGGGATCTGAGAGTGGACACAAAGTCCAGGGCCGCCTGGGCACGGCTCCTGCAAGAGCACCCTAACGCTCGGAAGTACAAAGGGGCCAATAAGAAGGGCCTCAGCAAAGGCTGCTTTGGCCTGAAACTGGACAGAATTGGCTCCATGTCCGGCCTCGGCTGCCCTTCCCTGCGGGACCCTCGGCCCAATGCCCCTTCCACTAGCGCTTAA-3′ (SEQ ID NO:10).

Spontaneously hypertensive rats (SHR) were divided into groups of sixrats each. One group included rats that were untreated, while the othergroups were treated with a single intravenous injection of AAV9 vectorsdesigned to express the rat proBNP polypeptide, the B-CDNP polypeptide,or the C-CDNP polypeptide. After five weeks, the rats were examined todetermine the weights of their hearts, lungs, and kidneys. Nosignificant difference was detected for the lungs and kidneys betweenthe treated and untreated animals (FIG. 7). There, however, was astatistically significant difference in the weight of the hearts for thetreated animals as compared to the weight of the hearts for theuntreated animals (FIG. 7). Administration of AAV9-C-CDNP facilitatedlong-term cardiac CDNP expression for five weeks in six treated rats ascompared to six untreated rats (FIG. 8).

These results demonstrate that a single intravenous administration of anatriuretic polypeptide-carrying AAV9 vector can protect the heart fromexcessive fibrosis and hypertophy in mammals with hypertension and/orwith renal dysfunction. These results also demonstrate that the methodsand materials provided herein can be used for long-term natriureticpolypeptide treatment (e.g., long-term CNDP treatment) of patients withcardiovascular and/or renal diseases (e.g., those patients with severecardiac hypertrophy and dysfunction).

In another set of experiments, an AAV9 vector designed to express BNPwas used to mediate cardiac BNP expression and extend the survival ofrats with established hypertensive heart disease (FIG. 10). Briefly, 9month-old SHR rats with established hypertensive heart disease weretreated with the AAV9 vector expressing rat proBNP.

Treated and untreated rats were monitored for survival. AAV9vector-mediated cardiac BNP expression extended the survival of ratswith established hypertensive heart disease (FIG. 10).

Sustained cardiac proBNP over-expression by the AAV9 vector improvedcardiac function and structure in established hypertensive heart disease(FIG. 11). Aged SHR rats (9 months old) with impaired cardiac functionswere randomly assigned to two groups, and 6 rats were intravenouslyinjected with proBNP-expressing AAV9 vector (1×10¹² genomeparticles/rat). The effects of sustained proBNP expression on cardiacfunctions/remodeling were analyzed at 5 months post injection. Whencompared to controls, septum wall thickness and LV mass weresignificantly reduced, while ejection fraction was significantly higherin BNP-treated rats (FIG. 11).

Expression of proBNP using an AAV9 vector designed to express proBNPresulted in improved cardiac functions in a rat model of polycystickidney disease (FIG. 13). Short-term (2 months) AAV9-proBNP treatmentsignificantly improved the cardiac function in a rat model of polycystickidney disease (PKD). Briefly, four week old PCK rats (a rat model ofPKD) were treated with the proBNP-encoding AAV9 vector. Two months aftersystemic vector administration, statistically significant improvementsin cardiac structure and functions (output) were observed (FIG. 13).

Example 3 Design of BNP and CDNP Viral Vectors for Human Use to TreatCardiovascular and/or Renal Diseases

To deliver human BNP to human cardiac cells, a cardio-tropic AAV9 vectorwas designed to express a human proBNP polypeptide having a humanN-terminus proBNP amino acid sequence (called an NT-proBNP region)upstream of a human BNP-32 amino acid sequence. The amino acid sequencefor the signal peptide and NT-proBNP region of the human proBNPpolypeptide wasMDPQTAPSRALLLLLFLHLAFLGGRSHPLGSPGSASDLETSGLQEQRNHLQGKLSELQVEQTSLEPLQESPRPTGVWKSREVATEGIRGHRKMVLYTLRAPR (SEQ ID NO:4). The amino acid sequence for the humanBNP-32 of the human proBNP polypeptide wasSPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH (SEQ ID NO:11). The amino acid sequencefor the human proBNP polypeptide wasMDPQTAPSRALLLLLFLHLAFLGGRSHPLGSPGSASDLETSGLQEQRNHLQGKLSELQVEQTSLEPLQESPRPTGVWKSREVATEGIRGHRKMVLYTLRAPRSPKMVQGSGCFGRKMDRISSSSGLGCKVLRRH (SEQ ID NO:12).

To deliver CDNP to human cardiac cells, two cardio-tropic AAV9 vectorswere designed. One AAV9 vector was designed to express a polypeptidedesignated human B-CDNP, and the other AAV9 vector was designed toexpress a polypeptide designated human C-CDNP. The human B-CDNPpolypeptide was designed to include a human N-terminus proBNP amino acidsequence (called an NT-proBNP region) upstream of the CDNP amino acidsequence, while the C-CDNP polypeptide was designed to include a humanN-terminus proCNP amino acid sequence (called an NT-proCNP region)upstream of the CDNP amino acid sequence. The amino acid sequence forthe human NT-proBNP region with signal peptide of the human B-CDNPpolypeptide wasMDPQTAPSRALLLLLFLHLAFLGGRSHPLGSPGSASDLETSGLQEQRNHLQGKLSELQVEQTSLEPLQESPRPTGVWKSREVATEGIRGHRKMVLYTLRAPR (SEQ ID NO:4), and the amino acidsequence for the human NT-proCNP region of the human C-CDNP polypeptidewasMHLSQLLACALLLTLLSLRPSEAKPGAPPKVPRTPPAEELAEPQAAGGGGKKGDKAPGGGGANLKGDRSRLLRDLRVDTKSRAAWARLLGEHPNARKYKGANKK (SEQ ID NO:5). The amino acid sequence for CDNP of thehuman B-CDNP and human C-CDNP polypeptides wasGLSKGCFGLKLDRIGSMSGLGCPSLRDPRPNAPSTSA (SEQ ID NO:6). The amino acidsequence for human B-CDNP polypeptide wasMDPQTAPSRALLLLLFLHLAFLGGRSHPLGSPGSASDLETSGLQEQRNHLQGKLSELQVEQTSLEPLQESPRPTGVWKSREVATEGIRGHRKMVLYTLRAPRGLSKGCFGLKLDRIGSMSGLGCPSLRDPRPNAPSTSA (SEQ ID NO:7), andthe amino acid sequence for human C-CDNP polypeptide wasMHLSQLLACALLLTLLSLRPSEAKPGAPPKVPRTPPAEELAEPQAAGGGQKKGDKAPGGGGANLKGDRSRLLRDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGCPSLRDPRPNAPSTSA (SEQ ID NO:8).

The nucleic acid sequences encoding human B-CDNP and human C-CDNP werecodon-optimized for expression in human cells. The nucleic acid sequenceencoding human B-CDNP was as follows5′-ATGGACCCACAGACAGCTCCCAGTAGGGCTTTGCTTCTTTTGCTTTTCCTGCACCTGGCTTTTCTGGGCGGACGATCCCATCCACTGGGTAGCCCTGGCTCCGCCTCAGATCTGGAGACTAGTGGACTGCAGGAGCAGCGCAATCACTTGCAGGGCAAACTGTCCGAGCTGCAGGTGGAACAAACGAGCCTCGAGCCCCTGCAGGAGAGCCCTAGACCTACCGGGGTGTGGAAGTCTCGAGAGGTAGCGACAGAAGGCATTAGAGGGCACAGGAAGATGGTACTGTATACTCTGAGGGCCCCAAGGGGACTGAGCAAGGGCTGTTTTGGCCTGAAGCTGGATCGGATTGGCAGCATGTCCGGCCTGGGCTGCCCTTCCCTGCGGGACCCACGGCCAAATGCCCCCTCCACCAGCGCCTAA-3′ (SEQ ID NO:9), while the nucleic acidsequence encoding human C-CDNP was as follows5′-ATGCATCTGTCCCAACTGCTGGCTTGTGCTCTCCTGCTGACTCTGCTGAGCCTCCGGCCTAGCGAGGCCAAGCCTGGAGCACCACCTAAGGTCCCCAGGACTCCTCCAGCCGAAGAACTGGCTGAGCCTCAGGCTGCCGGGGGCGGGCAGAAGAAAGGAGACAAAGCCCCTGGAGGGGGCGGGGCTAATCTCAAGGGCGATAGGTCCAGACTGCTGAGGGATCTGAGAGTGGACACAAAGTCCAGGGCCGCCTGGGCACGGCTCCTGCAAGAGCACCCTAACGCTCGGAAGTACAAAGGGGCCAATAAGAAGGGCCTCAGCAAAGGCTGCTTTGGCCTGAAACTGGACAGAATTGGCTCCATGTCCGGCCTCGGCTGCCCTTCCCTGCGGGACCCTCGGCCCAATGCCCCTTCCACTAGCGCTTAA-3′ (SEQ ID NO:10).

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. (canceled)
 2. A method for improving cardiac function in a mammalwith polycystic kidney disease, wherein said method comprisesadministering an AAV8 vector to said mammal with polycystic kidneydisease, wherein said AAV8 vector comprises a nucleic acid sequenceencoding a natriuretic polypeptide, and wherein cardiac function isimproved at least two months following said administration when saidAAV8 vector is administered to said mammal with polycystic kidneydisease.
 3. The method of claim 2, wherein said mammal is a human. 4.The method of claim 2, wherein said natriuretic polypeptide is a humanBNP polypeptide.
 5. The method of claim 2, wherein said natriureticpolypeptide is a CDNP polypeptide.
 6. The method of claim 2, whereinsaid natriuretic polypeptide is a B-CDNP polypeptide.
 7. The method ofclaim 2, wherein said natriuretic polypeptide is a C-CDNP polypeptide.